Optical microscopy has become an indispensable tool in research, clinical and industry labs. Contrast of imaging of optically transparent samples is limited when such imaging is carried out with traditional optical microscopy. To enhance imaging contrast, various strategies including fluorescence labeling have been developed. In comparison with imaging utilizing traditional optical microscopy, fluorescence imaging requires additional sample-preparation steps, which may distort the natural properties of the molecules of the object being imaged. Fluorescence imaging is also subject to blinking and photobleaching, which shortcomings make it difficult to quantify the image intensity and study single molecules over a long time. The term “blinking” as used herein refers to the phenomenon of random switching between ON (bright) and OFF (dark) states of lights emitters (such as, for example, molecular fluorophores, or colloidal quantum dots) associated with the object under the condition of continuous excitation. In addition, due to relatively weak fluorescence emission, the speed of the fluorescence imaging procedure is relatively slow, which is not suitable for imaging of fast biological processes.
The ability of imaging or otherwise visualizing single DNA molecules is critical for studying the biophysical and biochemical properties of DNA and for developing various applications utilizing such properties (such as, for example, sequencing DNA and studying DNA-protein interactions). An important example of such applications is a technique for obtaining, with the use of optical mapping, high-resolution genome-wide restriction maps of single DNA molecules (discussed, for example, by Teague, et al., in High-resolution human genome structure by single-molecule analysis. Proc Natl Acad Sci USA, 2010 107(24):10848-10853, which publication is incorporated by reference herein). The resulting maps serve as a “barcode” or “finger print” for the sequence of an unknown DNA of an organism. The current approach to the above-identified optical mapping relies on fluorescence microscopy, during which labeling of DNA with fluorescent dyes (such as YOYO-1, for example) is found to elongate and twist the native structure of the DNA, and affect the charge distribution of DNA. The optical mapping technique that is devoid of (or does not require) labeling DNA with fluorescent dye (a label-free technique) would facilitate the elimination of these effects, and provide additional information because such technique would facilitate measurement of the intrinsic physical characteristics of DNA instead of those of the labels. As used herein, the term “label-free technique” refers to a process of detection without the need to covalently attach a fluorophore to the molecule being detected (such as a protein or nucleic acid, for example). The atomic force microscopy (AFM) is a powerful label-free technique for imaging single DNA molecules, but it is operationally slow (taking seconds to minutes to acquire a single image). Additionally, the scanning of the AFM probe may perturb the DNA samples.
Surface plasmon resonance is another example of a label-free technique, which has been used to study molecular bindings. Recently, imaging of single viruses with high-resolution surface plasmon resonance microscopy has been demonstrated by Applicants (Wang, et al., Label-free imaging, detection, and mass measurement of single viruses by surface plasmon resonance. Proc Natl Acad Sci USA 107(37): 16028-16032, 2010). Despite of the advances, it remains a difficult challenge to image single molecules, such as DNA, proteins or any other biomolecules or macromolecules with surface plasmon resonance microscopy. A primary reason is the background noise associated with the surface plasmon resonance microscope, including interference patterns arising from the coherence of light, dirt on and imperfection of the optical components, including objective and light sources, and non-uniform distribution of the light illumination. The present invention discloses a differential imaging method and apparatus aiming at removing the background noise, thus allowing for label-free imaging of biological molecules.